Role of HNF4a in osteoblasts [ChIP-seq]

Murine osteoblast-like cell line MC3T3-E1 subclone 4 (ATCC CRL-2593) (ATTCC, Manassas, USA) were stably transfected using 4D-Nucleofector System (Lonza, Basel, Switzerland) to generate cells that overexpress Hnf4α1, Hnf4α2 or an empty vector (Ctr). ChIPseq analyzes were used to highlight the importance of HNF4a1 and HNF4a2 in osteoblastogenesis. We immunoprecipitated HNF4α in samples isolated from cells overexpressing Hnf4α1, Hnf4α2 or Ctr MC3T3 cultures using two different polyclonal antiHNF4α antibodies purchased from 1) Aviva or 2) Abcam. In parallel, we generated two stable cell lines overexpressing Hnf4α2 coupled with a carboxy (Hnf4α2C-Halo-Tg) or amino (Hnf4α2N-Halo-Tg) terminal Halo tag and used an anti-Halo antibody to IP these cells. Cells were cultured into osteoblastic medium for 21 days. Cell cultures were subjected to ChIP assay following the protocol provided by manufacturer (SimpleChIP Plus Enzymatic Chromatin IP Kit with magnetic beads, Cell Signaling Technology, Danvers MA, USA). Briefly, protein-chromatin crosslinking was carried out in cell medium containing 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 10 minutes. After stopping the crosslinking reaction using 10X glycine for 5 min, cells were washed 3 times in PBS and scraped into cold PBS containing protease inhibitor cocktail. Collected cells were centrifuged at 2000xg for 5 min at 4C. After nuclei extraction, chromatin was digested with micrococcal nuclease, and nuclear membranes were disrupted by sonication. Lysates were clarified by centrifugation at 9400xg for 10 min at 4C. Per IP reaction, 10 µg of digested, cross-linked chromatin were incubated with anti-HNF4α antibodies for immunoprecipitation: 5 µg of OASG03561 (Aviva Systems Biology, San Diego, CA, USA), 10 µg of ab41898 (Abcam, Cambridge, UK), or 10 µg of anti-Halotag antibody (G9281, Promega, Madison, WI, USA) for 4h at 4C. Then, 30 μl of Dynabeads Protein G Magnetic Beads was added to the IP chromatin solution and incubated for 2h at 4C. After several washing steps using buffers with ascending NaCl concentrations, chromatin was eluted, and the supernatant was incubated with proteinase K overnight at 65C to reverse crosslinking. Finally, DNA fragments were purified using silica columns. For each condition, we used three separate biological replicates and one input control. The total DNA library for each individual sample was prepared using the TruSeq ChIP-Seq Library Prep Kit (Illumina, San Diego, CA), and the bar-coded cDNA libraries were sequenced for 100 bp single reads using the Illumina Hiseq 4000. The ENCODE pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2) v 1.7.1 was used to identify naive overlapping peaks in each experiment. Enriched regions were consolidated based on their representation in two or more experiments as follow: HNF4α1/2 peaks if detected in samples expressing Hnf4α1 and Hnf4α2; HNF4α1 peaks if detected in two or more samples overexpressing Hnf4α1 or Ctr cells; HNF4α2 peaks if detected in two or more samples overexpressing Hnf4α2 or Ctr cells.